Our research focuses on ATP-dependent molecular chaperones, proteins that mediate protein folding, assembly, and disassembly without themselves being part of the final complexes. They participate in many cellular processes including DNA replication, regulation of gene expression, cell division, protection and recovery from stress conditions, and membrane translocation. They also participate directly in energy-dependent proteolysis. The high degree of conservation of the molecular chaperones and ATP-dependent proteases throughout evolution suggests that the fundamental mechanisms that we are defining by studying Escherichia coli proteins will lead to the discovery of similar roles for chaperones and proteases in regulating protein activity and degradation in other organisms. Since our initial discovery that Clp ATPases are molecular chaperones, we have studied various aspects of the mechanism of action of ClpA and ClpX in protein remoldeling and in degradation in conjunction with a proteolytic component, ClpP. We explored how ClpA recognizes specific substrates, since it had been impossible to identify a simple linear recognition motif by aligning sequences of known substrates. A site in a specific ClpA substrate, RepA, has been characterized that is responsible for the interaction of the substrate with ClpA. By analyzing RepA derivatives with N- or C-terminal deletions, we found that the N-terminal portion of RepA is required for recognition. More precisely, the signal resides near, but not at the N-terminus, in the vicinity of amino acids 10 to 15. We constructed fusions of RepA and green fluorescent protein (GFP), a protein not recognized by ClpA. Results from studies of these fusion proteins demonstrated that the N-terminal 15 amino acids of RepA are both necessary and sufficient to target the fusion protein for degradation by ClpAP in vitro and in vivo. We have studied the binding and unfolding of proteins by ClpA and ClpX and the translocation of unfolded proteins to ClpP in collaboration with M. Maurizi's Laboratory. ClpA and ClpX substrate recognition signals were fused to GFP and unfolding was measured by a disappearance of fluorescence. Both ClpA and ClpX catalyze ATP-dependent protein unfolding in the absence of ClpP. We found that although ClpA is unable to recognize native proteins lacking recognition signals, it binds untagged proteins when they are unfolded. Release of unfolded untagged proteins from ClpA or degradation by ClpAP requires ATP hydrolysis. Thus, both the initial unfolding step and the translocation step require ATP. Based on the high affinity interaction observed between unfolded proteins and ClpA in vitro, ClpA may play a role in vivo by interacting with unfolded proteins that escape surveillance by the predominant chaperones. We have visualized some of the interactions of ClpAP with RepA by cryoelectron microscopy, in collaboration with A. Steven's and M. Maurizi's laboratories. A RepA dimer is seen at a near axial site on the distal surface of ClpA relative to ClpP. When chemically inactivated ClpP is used, the addition of ATP results in the translocation of RepA into the digestion chamber of ClpP. Little change in ClpAP is observed, implying that translocation proceeds without major reorganization of ClpA. In collaboration with S. Gottesman's and M. Maurizi's laboratories, we have studied a ClpXP specificity enhancing factor, RssB. Degradation of sigma S, an RNA polymerase sigma factor that regulates expression of stationary phase and stress response genes, requires ClpXP together with RssB. Interestingly, RssB is homologous to response regulator proteins and its activity is regulated through specific signaling pathways. Using purified components, we reconstructed the degradation of Sigma S in vitro and demonstrated a direct role for RssB in delivering sigma S to ClpXP. RssB greatly stimulates sigma S degradation by ClpXP and acts catalytically. Acetyl phosphate, which phosphorylates RssB, is required. RssB promotes sigma S degradation specifically; it does not affect degradation of other ClpXP substrates or other proteins not normally degraded by ClpXP. Alone neither sigma S nor RssB binds ClpX with high affinity. Together, sigma S and RssB form a stable complex in the presence of acetyl phosphate, and in cooperation with ClpX, they form a ternary complex. When ClpP is present, a larger sigma S-RssB-ClpXP complex forms. The complex degrades sigma S and releases RssB from ClpXP in an ATP-dependent reaction. Thus, RssB is an example of a trans-targeting protein specifically enabling the degradation of sigma S by ClpXP by increasing the affinity of sigma S for ClpX.